Methotrexate is an antineoplastic and immunosuppressive agent useful, for example, in the chemotherapy treatment of leukemic meningitis, osteogenic sarcoma, psoriasis and other disorders and malignancies in animals and man. Methotrexate is an antagonist to folic acid.
The drug methotrexate competitively inhibits the enzyme dihydrofolate reductase. The conversion of dihydrofolate to tetrahydrofolate is necessary for the biosynthesis of thymidylic acid, a precursor of DNA. Inhibition of dihydrofolate reductase by methotrexate blocks the DNA synthetic pathway and is selectively lethal to rapidly dividing cells.
The use of methotrexate in systemic and intrathecal, as well as conventional and high-dose drugs, therapy requires that the drug be carefully monitored to avoid clinical toxicity to the patient, and to permit the introduction of a rescue agent like leucovorin before the critical toxicity level is reached. Methotrexate is eliminated by the patient with time; however, the elimination rate often varies markedly in some patients. Toxicity can arise from the delayed elimination of the drug by the patient. Thus, a rapid, simple and accurate technique and system for determining the level of methotrexate is important.
Present techniques for the monitoring of methotrexate in biological fluids are not wholly satisfactory, since such techniques are often complex, time-consuming and of limited availability. Some present suggested methods to assay methotrexate levels include: an enzyme-inhibition assay; radioimmunoassay, a flurorometrical method, and the use of a tritium radiolabeled drug.
One radioassay technique (see Raso and Schreiber, Cancer Research 35, 1407, June 1975) employs a tritium-labeled methotrexate, and uses a human serum buffer system with an enzyme from leukemia cells in a sequential assay technique, with the enzyme added first to the methotrexate sample.
Another radioassay technique, the Myers et al system (see Myers, Lippman, Eliot and Chabner, Proc. Nat. Acad. Sci. USA, Vol. 72, No. 9, pp. 3683-3686, September 1975, Medical Sciences), employs a competitive protein binding assay using tritium-labeled methotrexate, and uses a heparin buffer system and plasma samples.